Robust axis elongation by Nodal-dependent restriction of BMP signaling.

Embryogenesis results from the coordinated activities of different signaling pathways controlling cell fate specification and morphogenesis. In vertebrate gastrulation, both Nodal and BMP signaling play key roles in germ layer specification and morphogenesis, yet their interplay to coordinate embryo patterning with morphogenesis is still insufficiently understood. Here, we took a reductionist approach using zebrafish embryonic explants to study the coordination of Nodal and BMP signaling for embryo patterning and morphogenesis. We show that Nodal signaling triggers explant elongation by inducing mesendodermal progenitors but also suppressing BMP signaling activity at the site of mesendoderm induction. Consistent with this, ectopic BMP signaling in the mesendoderm blocks cell alignment and oriented mesendoderm intercalations, key processes during explant elongation. Translating these ex vivo observations to the intact embryo showed that, similar to explants, Nodal signaling suppresses the effect of BMP signaling on cell intercalations in the dorsal domain, thus allowing robust embryonic axis elongation. These findings suggest a dual function of Nodal signaling in embryonic axis elongation by both inducing mesendoderm and suppressing BMP effects in the dorsal portion of the mesendoderm.

Maternal TGF-ß ligand Panda breaks the radial symmetry of the sea urchin embryo by antagonizing the Nodal type II receptor ACVRII.

In the highly regulative embryo of the sea urchin Paracentrotus lividus, establishment of the dorsal-ventral (D/V) axis critically depends on the zygotic expression of the TGF-ß nodal in the ventral ectoderm. nodal expression is first induced ubiquitously in the 32-cell embryo and becomes progressively restricted to the presumptive ventral ectoderm by the early blastula stage. This early spatial restriction of nodal expression is independent of Lefty, and instead relies on the activity of Panda, a maternally expressed TGF-ß ligand related to Lefty and Inhibins, which is required maternally for D/V axis specification. However, the mechanism by which Panda restricts the early nodal expression has remained enigmatic and it is not known if Panda works like a BMP ligand by opposing Nodal and antagonizing Smad2/3 signaling, or if it works like Lefty by sequestering an essential component of the Nodal signaling pathway. In this study, we report that Panda functions as an antagonist of the TGF-ß type II receptor ACVRII (Activin receptor type II), which is the only type II receptor for Nodal signaling in the sea urchin and is also a type II receptor for BMP ligands. Inhibiting translation of acvrII mRNA disrupted D/V patterning across all 3 germ layers and caused acvrII morphants to develop with a typical Nodal loss-of-function phenotype. In contrast, embryos overexpressing acvrII displayed strong ectopic Smad1/5/8 signaling at blastula stages and developed as dorsalized larvae, a phenotype very similar to that caused by over activation of BMP signaling. Remarkably, embryos co-injected with acvrII mRNA and panda mRNA did not show ectopic Smad1/5/8 signaling and developed with a largely normal dorsal-ventral polarity. Furthermore, using an axis induction assay, we found that Panda blocks the ability of ACVRII to orient the D/V axis when overexpressed locally. Using co-immunoprecipitation, we showed that Panda physically interacts with ACVRII, as well as with the Nodal co-receptor Cripto, and with TBR3 (Betaglycan), which is a non-signaling receptor for Inhibins in mammals. At the molecular level, we have traced back the antagonistic activity of Panda to the presence of a single proline residue, conserved with all the Lefty factors, in the ACVRII binding motif of Panda, instead of a serine as in most of TGF-ß ligands. Conversion of this proline to a serine converted Panda from an antagonist that opposed Nodal signaling and promoted dorsalization to an agonist that promoted Nodal signaling and triggered ventralization when overexpressed. Finally, using phylogenomics, we analyzed the emergence of the agonist and antagonist form of Panda in the course of evolution. Our data are consistent with the idea that the presence of a serine at that position, like in most TGF-ß, was the ancestral condition and that the initial function of Panda was possibly in promoting and not in antagonizing Nodal signaling. These results highlight the existence of key functional and structural elements conserved between Panda and Lefty, allow to draw an intriguing parallel between sea urchin Panda and mammalian Inhibin α and raise the unexpected possibility that the original function of Panda may have been in activation of the Nodal pathway rather than in its inhibition.

Molecular analysis of a self-organizing signaling pathway for Xenopus axial patterning from egg to tailbud.

Xenopus embryos provide a favorable material to dissect the sequential steps that lead to dorsal-ventral (D-V) and anterior-posterior (A-P) cell differentiation. Here, we analyze the signaling pathways involved in this process using loss-of-function and gain-of-function approaches. The initial step was provided by Hwa, a transmembrane protein that robustly activates early ß-catenin signaling when microinjected into the ventral side of the embryo leading to complete twinned axes. The following step was the activation of Xenopus Nodal-related growth factors, which could rescue the depletion of ß-catenin and were themselves blocked by the extracellular Nodal antagonists Cerberus-Short and Lefty. During gastrulation, the Spemann-Mangold organizer secretes a cocktail of growth factor antagonists, of which the BMP antagonists Chordin and Noggin could rescue simultaneously D-V and A-P tissues in ß-catenin-depleted embryos. Surprisingly, this rescue occurred in the absence of any ß-catenin transcriptional activity as measured by ß-catenin activated Luciferase reporters. The Wnt antagonist Dickkopf (Dkk1) strongly synergized with the early Hwa signal by inhibiting late Wnt signals. Depletion of Sizzled (Szl), an antagonist of the Tolloid chordinase, was epistatic over the Hwa and Dkk1 synergy. BMP4 mRNA injection blocked Hwa-induced ectopic axes, and Dkk1 inhibited BMP signaling late, but not early, during gastrulation. Several unexpected findings were made, e.g., well-patterned complete embryonic axes are induced by Chordin or Nodal in ß-catenin knockdown embryos, dorsalization by Lithium chloride (LiCl) is mediated by Nodals, Dkk1 exerts its anteriorizing and dorsalizing effects by regulating late BMP signaling, and the Dkk1 phenotype requires Szl.

Nodal negatively regulates osteoclast differentiation by inducing STAT1 phosphorylation.

Several members of the transforming growth factor beta (TGF-ß) superfamily regulate the proliferation, differentiation, and function of bone-forming osteoblasts and bone-resorbing osteoclasts. However, it is still unknown whether Nodal, a member of the TGF-ß superfamily, serves a function in bone cells. In this study, we found that Nodal did not have any function in osteoblasts but instead negatively regulated osteoclast differentiation. Nodal inhibited RANKL-induced osteoclast differentiation by downregulating the expression of pro-osteoclastogenic genes, including c-fos, Nfatc1, and Blimp1, and upregulating the expression of antiosteoclastogenic genes, including Bcl6 and Irf8. Nodal activated STAT1 in osteoclast precursor cells, and STAT1 downregulation significantly reduced the inhibitory effect of Nodal on osteoclast differentiation. These findings indicate that Nodal activates STAT1 to downregulate or upregulate the expression of pro-osteoclastogenic or antiosteoclastogenic genes, respectively, leading to the inhibition of osteoclast differentiation. Moreover, the inhibitory effect of Nodal on osteoclast differentiation contributed to the reduction of RANKL-induced bone loss in vivo.

A network of transcription factors governs the dynamics of NODAL/Activin transcriptional responses.

SMAD2, an effector of the NODAL/Activin signalling pathway, regulates developmental processes by sensing distinct chromatin states and interacting with different transcriptional partners. However, the network of factors that controls SMAD2 chromatin binding and shapes its transcriptional programme over time is poorly characterised. Here, we combine ATAC-seq with computational footprinting to identify temporal changes in chromatin accessibility and transcription factor activity upon NODAL/Activin signalling. We show that SMAD2 binding induces chromatin opening genome wide. We discover footprints for FOXI3, FOXO3 and ZIC3 at the SMAD2-bound enhancers of the early response genes, Pmepa1 and Wnt3, respectively, and demonstrate their functionality. Finally, we determine a mechanism by which NODAL/Activin signalling induces delayed gene expression, by uncovering a self-enabling transcriptional cascade whereby activated SMADs, together with ZIC3, induce the expression of Wnt3. The resultant activated WNT pathway then acts together with the NODAL/Activin pathway to regulate expression of delayed target genes in prolonged NODAL/Activin signalling conditions. This article has an associated First Person interview with the first author of the paper.

TGFß family signaling and development.

The transforming growth factor ß (TGFß) signaling family is evolutionarily conserved in metazoans. The signal transduction mechanisms of TGFß family members have been expansively investigated and are well understood. During development and homeostasis, numerous TGFß family members are expressed in various cell types with temporally changing levels, playing diverse roles in embryonic development, adult tissue homeostasis and human diseases by regulating cell proliferation, differentiation, adhesion, migration and apoptosis. Here, we discuss the molecular mechanisms underlying signal transduction and regulation of the TGFß subfamily pathways, and then highlight their key functions in mesendoderm induction, dorsoventral patterning and laterality development, as well as in the formation of several representative tissues/organs.

Deciphering and modelling the TGF-ß signalling interplays specifying the dorsal-ventral axis of the sea urchin embryo.

During sea urchin development, secretion of Nodal and BMP2/4 ligands and their antagonists Lefty and Chordin from a ventral organiser region specifies the ventral and dorsal territories. This process relies on a complex interplay between the Nodal and BMP pathways through numerous regulatory circuits. To decipher the interplay between these pathways, we used a combination of treatments with recombinant Nodal and BMP2/4 proteins and a computational modelling approach. We assembled a logical model focusing on cell responses to signalling inputs along the dorsal-ventral axis, which was extended to cover ligand diffusion and enable multicellular simulations. Our model simulations accurately recapitulate gene expression in wild-type embryos, accounting for the specification of ventral ectoderm, ciliary band and dorsal ectoderm. Our model simulations further recapitulate various morphant phenotypes, reveal a dominance of the BMP pathway over the Nodal pathway and stress the crucial impact of the rate of Smad activation in dorsal-ventral patterning. These results emphasise the key role of the mutual antagonism between the Nodal and BMP2/4 pathways in driving early dorsal-ventral patterning of the sea urchin embryo.

Blastocyst-like structures generated solely from stem cells.

The blastocyst (the early mammalian embryo) forms all embryonic and extra-embryonic tissues, including the placenta. It consists of a spherical thin-walled layer, known as the trophectoderm, that surrounds a fluid-filled cavity sheltering the embryonic cells 1 . From mouse blastocysts, it is possible to derive both trophoblast 2 and embryonic stem-cell lines 3 , which are in vitro analogues of the trophectoderm and embryonic compartments, respectively. Here we report that trophoblast and embryonic stem cells cooperate in vitro to form structures that morphologically and transcriptionally resemble embryonic day 3.5 blastocysts, termed blastoids. Like blastocysts, blastoids form from inductive signals that originate from the inner embryonic cells and drive the development of the outer trophectoderm. The nature and function of these signals have been largely unexplored. Genetically and physically uncoupling the embryonic and trophectoderm compartments, along with single-cell transcriptomics, reveals the extensive inventory of embryonic inductions. We specifically show that the embryonic cells maintain trophoblast proliferation and self-renewal, while fine-tuning trophoblast epithelial morphogenesis in part via a BMP4/Nodal-KLF6 axis. Although blastoids do not support the development of bona fide embryos, we demonstrate that embryonic inductions are crucial to form a trophectoderm state that robustly implants and triggers decidualization in utero. Thus, at this stage, the nascent embryo fuels trophectoderm development and implantation.

Self-organization of a human organizer by combined Wnt and Nodal signalling.

In amniotes, the development of the primitive streak and its accompanying 'organizer' define the first stages of gastrulation. Although these structures have been characterized in detail in model organisms, the human primitive streak and organizer remain a mystery. When stimulated with BMP4, micropatterned colonies of human embryonic stem cells self-organize to generate early embryonic germ layers 1 . Here we show that, in the same type of colonies, Wnt signalling is sufficient to induce a primitive streak, and stimulation with Wnt and Activin is sufficient to induce an organizer, as characterized by embryo-like sharp boundary formation, markers of epithelial-to-mesenchymal transition and expression of the organizer-specific transcription factor GSC. Moreover, when grafted into chick embryos, human stem cell colonies treated with Wnt and Activin induce and contribute autonomously to a secondary axis while inducing a neural fate in the host. This fulfils the most stringent functional criteria for an organizer, and its discovery represents a milestone in human embryology.

The SMAD2/3 interactome reveals that TGFß controls m6A mRNA methylation in pluripotency.

The TGFß pathway has essential roles in embryonic development, organ homeostasis, tissue repair and disease. These diverse effects are mediated through the intracellular effectors SMAD2 and SMAD3 (hereafter SMAD2/3), whose canonical function is to control the activity of target genes by interacting with transcriptional regulators. Therefore, a complete description of the factors that interact with SMAD2/3 in a given cell type would have broad implications for many areas of cell biology. Here we describe the interactome of SMAD2/3 in human pluripotent stem cells. This analysis reveals that SMAD2/3 is involved in multiple molecular processes in addition to its role in transcription. In particular, we identify a functional interaction with the METTL3-METTL14-WTAP complex, which mediates the conversion of adenosine to N6-methyladenosine (m6A) on RNA. We show that SMAD2/3 promotes binding of the m6A methyltransferase complex to a subset of transcripts involved in early cell fate decisions. This mechanism destabilizes specific SMAD2/3 transcriptional targets, including the pluripotency factor gene NANOG, priming them for rapid downregulation upon differentiation to enable timely exit from pluripotency. Collectively, these findings reveal the mechanism by which extracellular signalling can induce rapid cellular responses through regulation of the epitranscriptome. These aspects of TGFß signalling could have far-reaching implications in many other cell types and in diseases such as cancer.

Establishment of Cardiac Laterality.

Formation of the vertebrate heart with its complex arterial and venous connections is critically dependent on patterning of the left-right axis during early embryonic development. Abnormalities in left-right patterning can lead to a variety of complex life-threatening congenital heart defects. A highly conserved pathway responsible for left-right axis specification has been uncovered. This pathway involves initial asymmetric activation of a nodal signaling cascade at the embryonic node, followed by its propagation to the left lateral plate mesoderm and activation of left-sided expression of the Pitx2 transcription factor specifying visceral organ asymmetry. Intriguingly, recent work suggests that cardiac laterality is encoded by intrinsic cell and tissue chirality independent of Nodal signaling. Thus, Nodal signaling may be superimposed on this intrinsic chirality, providing additional instructive cues to pattern cardiac situs. The impact of intrinsic chirality and the perturbation of left-right patterning on myofiber organization and cardiac function warrants further investigation. We summarize recent insights gained from studies in animal models and also some human clinical studies in a brief overview of the complex processes regulating cardiac asymmetry and their impact on cardiac function and the pathogenesis of congenital heart defects.

Tril dampens Nodal signaling through Pellino2- and Traf6-mediated activation of Nedd4l.

Toll-like receptor 4 (Tlr) interactor with leucine-rich repeats (Tril) functions as a Tlr coreceptor to mediate innate immunity in adults. In Xenopus embryos, Tril triggers degradation of the transforming growth factor ß (Tgf-ß) family inhibitor, Smad7. This enhances bone morphogenetic protein (Bmp) signaling to enable ventral mesoderm to commit to a blood fate. Here, we show that Tril simultaneously dampens Nodal signaling by catalytically activating the ubiquitin ligase NEDD4 Like (Nedd4l). Nedd4l then targets Nodal receptors for degradation. How Tril signals are transduced in a nonimmune context is unknown. We identify the ubiquitin ligase Pellino2 as a protein that binds to the cytoplasmic tail of Tril and subsequently forms a complex with Nedd4l and another E3 ligase, TNF-receptor associated factor 6 (Traf6). Pellino2 and Traf6 are essential for catalytic activation of Nedd4l, both in Xenopus and in mammalian cells. Traf6 ubiquitinates Nedd4l, which is then recruited to membrane compartments where activation occurs. Collectively, our findings reveal that Tril initiates a noncanonical Tlr-like signaling cascade to activate Nedd4l, thereby coordinately regulating the Bmp and Nodal arms of the Tgf-ß superfamily during vertebrate development.

Role of EBAF/Nodal/p27 signaling pathway in development of placenta in normal and diabetic rats.

Placentas control the maternal-fetal transport of nutrients and gases. Placental reactions to adverse intrauterine conditions affect fetal development. Such adverse conditions occur in pregnancies complicated by diabetes, leading to alterations in placental anatomy and physiology. In this study, streptozocin (STZ) injection produced sustained hyperglycemia during pregnancy in rats. Hyperglycemic pregnant rats had gained significantly less weight than normal pregnant rats on embryonic day 15.5. We investigated the influence of diabetes on placental anatomy and physiology. Compared with controls, the diabetic group had a markedly thicker junctional zone at embryonic day 15.5. To explore a mechanism for this abnormality, we examined Nodal expression in the junctional zone of control and diabetic groups. We found lower expression of Nodal in the diabetic group. We then investigated the expression of its target gene p27Kip1 (p27), which is related to cell proliferation. In vitro, Nodal overexpression up-regulated p27 protein levels while interfered EBAF up-regulated p27. In vivo, the expression of p27 was lower in diabetic compared with normal rats, and localization was similar between the two groups. In contrast, a higher expression of PCNA was found in diabetic versus normal placenta. Endometrial bleeding associated factor (EBAF), an up-stream molecular regulator of Nodal, was expressed at higher levels in placenta from diabetic versus normal rats. Based on these results, we speculate that the EBAF/Nodal/p27 signaling pathway plays a role in morphological change of diabetic placenta.

Chronic demyelination and myelin repair after spinal cord injury in mice: A potential link for glutamatergic axon activity.

Our prior work examining endogenous repair after spinal cord injury (SCI) in mice revealed that large numbers of new oligodendrocytes (OLs) are generated in the injured spinal cord, with peak oligodendrogenesis between 4 and 7 weeks post-injury (wpi). We also detected new myelin formation over 2 months post-injury (mpi). Our current work significantly extends these results, including quantification of new myelin through 6 mpi and concomitant examination of indices of demyelination. We also examined electrophysiological changes during peak oligogenesis and a potential mechanism driving OL progenitor cell (OPC) contact with axons. Results reveal peak in remyelination occurs during the 3rd mpi, and that myelin generation continues for at least 6 mpi. Further, motor evoked potentials significantly increased during peak remyelination, suggesting enhanced axon potential conduction. Interestingly, two indices of demyelination, nodal protein spreading and Nav1.2 upregulation, were also present chronically after SCI. Nav1.2 was expressed through 10 wpi and nodal protein disorganization was detectable throughout 6 mpi suggesting chronic demyelination, which was confirmed with EM. Thus, demyelination may continue chronically, which could trigger the long-term remyelination response. To examine a potential mechanism that may initiate post-injury myelination, we show that OPC processes contact glutamatergic axons in the injured spinal cord in an activity-dependent manner. Notably, these OPC/axon contacts were increased 2-fold when axons were activated chemogenetically, revealing a potential therapeutic target to enhance post-SCI myelin repair. Collectively, results show the surprisingly dynamic nature of the injured spinal cord over time and that the tissue may be amenable to treatments targeting chronic demyelination.

A Nodal/Eph signalling relay drives the transition from apical constriction to apico-basal shortening in ascidian endoderm invagination.

Gastrulation is the first major morphogenetic event during animal embryogenesis. Ascidian gastrulation starts with the invagination of 10 endodermal precursor cells between the 64- and late 112-cell stages. This process occurs in the absence of endodermal cell division and in two steps, driven by myosin-dependent contractions of the acto-myosin network. First, endoderm precursors constrict their apex. Second, they shorten apico-basally, while retaining small apical surfaces, thereby causing invagination. The mechanisms that prevent endoderm cell division, trigger the transition between step 1 and step 2, and drive apico-basal shortening have remained elusive. Here, we demonstrate a conserved role for Nodal and Eph signalling during invagination in two distantly related ascidian species, Phallusia mammillata and Ciona intestinalis Specifically, we show that the transition to step 2 is triggered by Nodal relayed by Eph signalling. In addition, our results indicate that Eph signalling lengthens the endodermal cell cycle, independently of Nodal. Finally, we find that both Nodal and Eph signals are dispensable for endoderm fate specification. These results illustrate commonalities as well as differences in the action of Nodal during ascidian and vertebrate gastrulation.

Functional evidence that Activin/Nodal signaling is required for establishing the dorsal-ventral axis in the annelid Capitella teleta.

The TGF-ß superfamily comprises two distinct branches: the Activin/Nodal and BMP pathways. During development, signaling by this superfamily regulates a variety of embryological processes, and it has a conserved role in patterning the dorsal-ventral body axis. Recent studies show that BMP signaling establishes the dorsal-ventral axis in some mollusks. However, previous pharmacological inhibition studies in the annelid Capitella teleta, a sister clade to the mollusks, suggests that the dorsal-ventral axis is patterned via Activin/Nodal signaling. Here, we determine the role of both the Activin/Nodal and BMP pathways as they function in Capitella axis patterning. Antisense morpholino oligonucleotides were targeted to Ct-Smad2/3 and Ct-Smad1/5/8, transcription factors specific to the Activin/Nodal and BMP pathways, respectively. Following microinjection of zygotes, resulting morphant larvae were scored for axial anomalies. We demonstrate that the Activin/Nodal pathway of the TGF-ß superfamily, but not the BMP pathway, is the primary dorsal-ventral patterning signal in Capitella These results demonstrate variation in the molecular control of axis patterning across spiralians, despite sharing a conserved cleavage program. We suggest that these findings represent an example of developmental system drift.

A chordate species lacking Nodal utilizes calcium oscillation and Bmp for left-right patterning.

Larvaceans are chordates with a tadpole-like morphology. In contrast to most chordates of which early embryonic morphology is bilaterally symmetric and the left-right (L-R) axis is specified by the Nodal pathway later on, invariant L-R asymmetry emerges in four-cell embryos of larvaceans. The asymmetric cell arrangements exist through development of the tailbud. The tail thus twists 90° in a counterclockwise direction relative to the trunk, and the tail nerve cord localizes on the left side. Here, we demonstrate that larvacean embryos have nonconventional L-R asymmetries: 1) L- and R-cells of the two-cell embryo had remarkably asymmetric cell fates; 2) Ca2+ oscillation occurred through embryogenesis; 3) Nodal, an evolutionarily conserved left-determining gene, was absent in the genome; and 4) bone morphogenetic protein gene (Bmp) homolog Bmp.a showed right-sided expression in the tailbud and larvae. We also showed that Ca2+ oscillation is required for Bmp.a expression, and that BMP signaling suppresses ectopic expression of neural genes. These results indicate that there is a chordate species lacking Nodal that utilizes Ca2+ oscillation and Bmp.a for embryonic L-R patterning. The right-side Bmp.a expression may have arisen via cooption of conventional BMP signaling in order to restrict neural gene expression on the left side.

Activin/nodal signaling and NANOG orchestrate human embryonic stem cell fate decisions by controlling the H3K4me3 chromatin mark.

Stem cells can self-renew and differentiate into multiple cell types. These characteristics are maintained by the combination of specific signaling pathways and transcription factors that cooperate to establish a unique epigenetic state. Despite the broad interest of these mechanisms, the precise molecular controls by which extracellular signals organize epigenetic marks to confer multipotency remain to be uncovered. Here, we use human embryonic stem cells (hESCs) to show that the Activin-SMAD2/3 signaling pathway cooperates with the core pluripotency factor NANOG to recruit the DPY30-COMPASS histone modifiers onto key developmental genes. Functional studies demonstrate the importance of these interactions for correct histone 3 Lys4 trimethylation and also self-renewal and differentiation. Finally, genetic studies in mice show that Dpy30 is also necessary to maintain pluripotency in the pregastrulation embryo, thereby confirming the existence of similar regulations in vivo during early embryonic development. Our results reveal the mechanisms by which extracellular factors coordinate chromatin status and cell fate decisions in hESCs.

Differential impact of TGFß/SMAD signaling activity elicited by Activin A and Nodal on endoderm differentiation of epiblast stem cells.

Allocation of cells to an endodermal fate in the gastrulating embryo is driven by Nodal signaling and consequent activation of TGFß pathway. In vitro methodologies striving to recapitulate the process of endoderm differentiation, however, use TGFß family member Activin in place of Nodal. This is despite Activin not known to have an in vivo role in endoderm differentiation. In this study, five epiblast stem cell lines were subjected to directed differentiation using both Activin A and Nodal to induce endodermal fate. A reporter line harboring endoderm markers FoxA2 and Sox17 was further analyzed for TGFß pathway activation and WNT response. We demonstrated that Activin A-treated cells remain more primitive streak-like when compared to Nodal-treated cells that have a molecular profile suggestive of more advanced differentiation. Activin A elicited a robust TGFß/SMAD activity, enhanced WNT signaling activity and promoted the generation of DE precursors. Nodal treatment resulted in lower TGFß/SMAD activity, and a weaker, sustained WNT response, and ultimately failed to upregulate endoderm markers. This is despite signaling response resembling more closely the activity seen in vivo. These findings emphasize the importance of understanding the downstream activities of Activin A and Nodal signaling in directing in vitro endoderm differentiation of primed-state epiblast stem cells.

Proteomic analysis of the IPF mesenchymal progenitor cell nuclear proteome identifies abnormalities in key nodal proteins that underlie their fibrogenic phenotype.

IPF is a progressive fibrotic lung disease whose pathogenesis remains incompletely understood. We have previously discovered pathologic mesenchymal progenitor cells (MPCs) in the lungs of IPF patients. IPF MPCs display a distinct transcriptome and create sustained interstitial fibrosis in immune deficient mice. However, the precise pathologic alterations responsible for this fibrotic phenotype remain to be uncovered. Quantitative mass spectrometry and interactomics is a powerful tool that can define protein alterations in specific subcellular compartments that can be implemented to understand disease pathogenesis. We employed quantitative mass spectrometry and interactomics to define protein alterations in the nuclear compartment of IPF MPCs compared to control MPCs. We identified increased nuclear levels of PARP1, CDK1, and BACH1. Interactomics implicated PARP1, CDK1, and BACH1 as key hub proteins in the DNA damage/repair, differentiation, and apoptosis signaling pathways respectively. Loss of function and inhibitor studies demonstrated important roles for PARP1 in DNA damage/repair, CDK1 in regulating IPF MPC stemness and self-renewal, and BACH1 in regulating IPF MPC viability. Our quantitative mass spectrometry studies combined with interactomic analysis uncovered key roles for nuclear PARP1, CDK1, and BACH1 in regulating IPF MPC fibrogenicity.